Protein degradation by proteasomes plays an essential role in the proliferation of malignant cells. The proteasome inhibitor VELCADE (bortezomib, PS-341) is being used for the treatment of multiple myeloma and is in clinical trials for the treatment of other cancers. The proteasome has three types of active sites: chymotrypsin-like (Beta 5), trypsin-like (Beta 2), and caspase-like (Beta 1). Our long-term goal is to understand the precise roles of these active sites as targets of anti-neoplastic agents and to use this information to design new, more potent, less toxic drugs of this class. Bortezomib was developed as an inhibitor of the Beta 5 site, which has long been considered the only suitable target for inhibition. However, bortezomib also inhibits the (Beta 1 site, and we have recently obtained data showing that inhibition of the Beta 1 site is needed to achieve maximal cytotoxicity. Based on these data and on critical review of the literature, we hypothesize that (a) all three active sites are drug molecular targets, (b) inhibition of at least two sites is required to achieve optimal cytotoxicity, (c) the therapeutic windows of proteasome inhibitors depend on which active sites they target, and (d) the exact pathways by which proteasome inhibitors induce apoptosis in malignant cells depend on which active sites they target. We will test this hypothesis in multiple myeloma and breast cancer cells using unique, cell-permeable, specific inhibitors of proteasome catalytic sites that we have developed and are developing. The specific aims of this proposal are as follows: (1) To determine the growth inhibitory and cytotoxic effects of specific inhibitors of (Beta 5, Beta1, and Beta 2 catalytic sites, alone and in combination with each other, on cells derived from multiple myeloma and breast cancers, on immortalized and transformed human mammary epithelial cells, and on human peripheral blood lymphocytes;(2) To determine whether the mechanisms by which proteasome inhibitors kill multiple myeloma cells depend on the catalytic sites being targeted by these agents;The results of these studies will define what active-site pharmacological specificities proteasome inhibitors must have to achieve optimal anti-neoplastic activity and therapeutic windows. Taken together, these studies will provide a strong rationale for development of novel inhibitors with desired molecular pharmacological properties. Such compounds have potentially broad applicability for cancer therapy.